Date of Award

2018

Embargo Period

8-1-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Neuroscience

College

College of Graduate Studies

First Advisor

Andy Shih

Second Advisor

DeAnna Adkins

Third Advisor

Peter Kalivas

Fourth Advisor

Tatyana Gudz

Fifth Advisor

Craig Brown

Abstract

The goal of this research is to investigate the role of pericytes during ischemia, and more specifically, to investigate their cell-specific proteolytic contributions that lead to the degeneration of the blood-brain barrier (BBB). BBB degeneration represents a pathological hallmark of ischemic stroke and is a major factor in limiting the window for pharmacological treatments by increasing the likelihood for hemorrhagic transformation. By understanding cell-specific proteolytic contributions to BBB degeneration during ischemia, novel biomarkers to predict the severity of the injury as well as new targets to reduce the extent of damage from stroke could be discovered. Pericytes are a cell intricately linked with the microvasculature and are necessary for the development and maintenance of the blood-brain barrier (BBB). While it is known that genetic ablation of pericytes leads to increased cerebrovascular leakage and buildup of neurotoxic molecules within the brain, the consequence of pericyte pathology in vivo remains poorly understood. In our experiments we used in vivo two-photon imaging and photothrombotic occlusions within the capillary bed to explore pericyte responses to ischemia. We have also developed a novel application of a quenched (FITC) gelatin probe (FITC-Gelatin), which rapidly increases several hundred-fold in fluorescence upon gelatinolytic cleavage performed by matrix metalloproteinases (MMP). This probe allows for in vivo two-photon imaging of MMP 2/9 activity with resolution at a cellular level. We also implement numerous pharmacological agents in order to determine the biochemical contributions to pericyte pathology during ischemia. We find that pericytes respond to ischemia with robust, locally active, MMP 9 which produces BBB degeneration at the location of pericyte somata. We have also found that the MMP activity occurs within tens of minutes following the induction of ischemia within the capillary bed and the cessation of blood flow is necessary for leakage to occur. In addition we have found that the initial activation of MMP 9 can be blocked through the inhibition of nitric oxide production (via L-NIL), and that this blockage occurs in a pericyte specific manner. From this we conclude that pericytes are responsible for rapid BBB degeneration during ischemia mediated through a post-translational activation of MMP 9 by NO.

Rights

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